Bone is a composite matrix composed of mineralized and aligned collagen nanofibers. Combination of inorganic apatite nanocrystals and organic collagen fibers provides bone with unique mechanical and biological properties. The apatite nanocrystals provide osteoconductivity and compressive strength while the collagen fibers provide elasticity and a template for mineralization and maturation of osteoprogenitor cells. Unique factors that contribute to bone toughness are the aligned network of collagen fibers, apatite nanocrystals, and proteins in the bone extracellular matrix (ECM) that link the apatite crystals to the collagen fibers. On a larger scale, laminated multilayers of calcium phosphate (CaP)-deposited aligned fibers form the cortical bone that is composed of osteons having microtube-like structures surrounding a central micro-canal that provides nutrient/waste transport to and from the bone tissue.
In an effort to mimic the natural morphology at the ECM level, electrospinning has been used to produce aligned nanofibers from natural biopolymers, like collagen and chitosan, or synthetic polymers such as poly(L-lactide) (PLLA) and poly(ε-Caprolactone) (PCL). Due to their nano-scale size and alignment, electrospun nanofibers provide enormous surface area for cell adhesion, migration, and differentiation, as well as deposition of bioactive agents.
Different methods have been used to create composites of nanofibers reinforced with CaP crystals to improve mechanical strength of the synthetics and provide a conductive matrix for osteoprogenitor cells. In one approach, CaP nanocrystals were mixed with the spinning solution and the solution was electrospun to form CaP composite nanofibers. In that approach, CaP loading and strength of the composite were limited by viscosity of the spinning solution. In another approach, electrospun nanofibers were laminated with a CaP paste to form a composite sheet. This approach was limited to use of the CaP paste however, and toughness of the composite depended on the extent of penetration of the paste into the fiber mesh. In a biomimetic approach, nanofibers were coated with CaP crystals by incubation in a modified simulated body fluid (SBF). This approach mimicked the morphology of the mineralized bone matrix but drawbacks included diffusion-limited penetration of calcium and phosphate ions in the central part of the fiber sheet and lack of crystal nucleation from the fiber surface as opposed to crystal nucleation in solution followed by deposition on the fiber surface. In another approach, a continuous uninterrupted layer of CaP crystals was deposited on the surface of nanofibers within an electric field. This approach produced CaP coated nanofibers at high deposition rate and CaP to fiber ratios exceeding 250% but the CaP layer was continuous and the CaP crystals were not covalently attached to the fiber surface.
What is needed in the art is a method for developing composites that are more accurate bone tissue biomimetics with high stiffness and interconnected microtubular structures to support the exchange of nutrients and oxygen. Ideally, these ECM-level biomimetics can then be used to create larger scale bone graft materials.
Cranial, maxillofacial, oral fractures and large bone defects are currently being treated by using auto- and allografts. Unfortunately, these grafts have limitations in clinical usage such as immune response, donor-site morbidity, and lack of availability. As a result, interest in tissue engineering materials and methods for bone graft procedures has rapidly been growing in an attempt to develop engineered bone grafts that can mimic the bone microstructure.
Tissue engineering approaches require a resilient cell supporting scaffold in order to maintain a 3-dimensional substrate for cell growth and development during the formation of bone tissue. The physical configurations of the scaffolds, which mediate the cell-cell and cell-scaffold interactions, exert strong influence on the success of osteogenic processes in vitro. The success of an engineered scaffold mostly depends on how closely the cell-scaffold relationship mimics that of natural tissue in vivo. Nanofiber composites such as those mentioned above have been used in an attempt to fabricate larger osteoinductive and/or osteoconductive scaffolding. Both in vitro and in vivo studies have demonstrated that organic/inorganic composite fibrous scaffolds support attachment, differentiation, and proliferation of osteoblasts or multipotent stromal cells (MSCs) and facilitate bone healing. However, investigations regarding the effect of fibrous composite scaffolds are still limited.
What are needed in the art are bone tissue biomimetic materials and methods that can be utilized to form rigid constructs in tissue engineering applications for the development of three dimensional mineralized and vascularized cellular structures, for instance in the formation of bone tissue biomimetic materials for use in bone graft applications.